In resting leukocytes, B2 integrin molecules are immobilized on the cell membrane in a diffused distribution, because they are constrained by cytoskeletal complex. Activation signals through the "inside-out" pathway cause this cytoskeletal complex to dissociate, resulting in enhanced free motion and clustering of f32 integrin. The clustered integrin provides high avidity binding to the ligands. The goal of this proposal is to identify and characterize the cytoskeletal complex constraining the integrin molecules in resting leukocytes, termed "preactivation cytoskeletal complex" in this proposal. Using the ability of regulating molecular mobility and clustering of B2 integrin as the criteria, we propose to determine whether talin, calmodulin, and dynamitin - three proteins involved in integrin activation-are members of the preactivation complex. Since integrin activation should not be a "none" or "all" process, two quantitative assays will be introduced: the single particle tracking method to quantitate the molecular mobility of B2 integrin and the fluorescent resonance energy transfer method to quantitate the clustering. Once we have identified the new members of the preactivation complex, their interrelationship with the currently known members, i.e. PKC, MacMARCKS, actin, and microtubule filaments, and among themselves will be determined by individually disrupting the function of each one by mutagenesis or by specific inhibitors. The significance of these studies is two-fold: if these proteins are indeed members of the preactivation complex, then the currently understood sequence of integrin activation is confirmed and we will have found new members. On the other hand, if these proteins regulate integrin activation without changing integrin mobility and clustering, then the currently held scheme of integrin activation requires modification. Information obtained from this study will provide a new understanding of the role of cytoskeletal proteins in regulating integrin activation. Since B2 integrin is a crucial regulator of the immune function in health and in diseases, this proposal has broad significance for clinical medicine.